The present invention relates to microwave assisted chemistry and in particular relates to the chemistry of peptide synthesis, specifically the chemistry of the protection and deprotection steps in solid phase peptide synthesis (“SPPS”).
A general background discussion of peptide synthesis, solid phase peptide synthesis and microwave assisted solid phase peptide synthesis is set forth in commonly assigned United States Patent Application Publication No. 20040260059, the contents of which are incorporated entirely herein by reference. Microwave enhancement of solid phase peptide synthesis is also commercially available in the form of the LIBERTY™ and DISCOVER® instruments available from CEM Corporation of Matthews, N.C., USA.
As further background, both three-letter and one-letter abbreviations are commonly used in this field to represent the 20 amino acids commonly found in proteins. In turn, groups or strings of these abbreviations are used to represent peptide chains. These abbreviations are widely used and recognized and will be understood in context when they appear herein.
As another detail, some sources prefer to refer to peptides as “polypeptides,” and it will be understood that these terms are equivalent. The term peptide is used predominantly herein.
As set forth in the '059 publication and as well understood in this art, solid phase peptide synthesis is typically carried out by adding a first amino acid to a solid phase resin particle and then adding second and successive acids to the first acid to form the peptide chain. Attaching the growing chain to the solid phase resin particles permits the step-wise reactions to be carried out more easily.
In order to prevent undesired reactions at inappropriate times, each amino acid typically includes an intentionally-added composition referred to as a “protecting group” on the amino side of the molecule. Adding a second amino acid to the first amino acid accordingly requires removing the protecting group from the first acid. This step is referred to as “deprotection.” Similarly (and successively), after the second acid and its protecting group have been added to the first acid, the protecting group must be removed from the second acid in order to add the third acid, and so forth.
As further known to those of skill in this art, the 9-fluorenylmethyloxycarbonyl group, commonly referred to as “N-Fmoc” (or simply “Fmoc”) is a favored composition for acting as the protecting group in peptide synthesis, including (but not limited to) SPPS. Accordingly, the step of removing the Fmoc (deprotection) must be carried out repeatedly; i.e., every time another protected acid is added to the growing peptide chain.
During SPPS, the N-Fmoc protecting group can be removed by organic bases in a base-catalyzed elimination. Deprotection is most efficient with unhindered secondary amines, but is also susceptible to primary and tertiary amines. Typically a solution of 20% piperidine in DMF (N,N-dimethly formamide) is used to form a dibenzofulvene (DBF) intermediate that is immediately trapped by the secondary amine to form an inert adduct.
For example, microwave-enchanced Fmoc deprotection was reported with 65-74ACP peptide. In this study complete deprotection was observed with 20% Piperidine in DMF in 1 minute. Without microwave multiple deletions were observed.
Piperidine has a favorable pKa (11.1), but because it is also a precursor for the synthesis of phenylcyclidine (“angel dust”) it is regulated (in the U.S.) by the Drug Enforcement Agency. Piperidine is also toxic by ingestion and a strong irritant.
In difficult peptides incomplete Fmoc deprotection can be a problem and the use of the stronger tertiary base, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) has been shown to increase reaction efficiency. Typically, small amounts of piperidine are added to DBU to scavenge the free DBF because DBU alone does not accomplish this. If not scavenged, free DBF can potentially react with the resulting resin-bound terminal Nα-amine preventing acylation of the next amino acid.
The deprotection reaction is often performed in two stages to prevent such DBF alkylation of the resin-bound terminal Nα-amine. The first stage is typically shorter than the second and serves to remove a significant amount of DBU from the reaction vessel before a longer deprotection reaction is employed with fresh reagent.
Base catalyzed aspartimide formation (described in detail in the literature) also can be a serious problem during peptide synthesis chain assembly. In this side reaction the nitrogen atom attached to the α-carboxy group of either aspartic acid or asparagine attacks the side chain ester or amide group, respectively. Nucleophilic attack then causes subsequent ring opening, which gives rise to a mixture of α-aspartyl and β-aspartyl peptides.
Aspartimide formation has been shown to occur in sequences containing the “Asp-X” moiety, where X=[Gly, Asn, Ser, Thr]. This sequence is diagrammed in FIG. 4. Each subsequent deprotection cycle after the “Asp-X” sequence further increases aspartimide formation. This can be a serious problem in longer peptides with multiple Asp residues.
This process naturally occurs in biological systems with proteins containing aspartic acids. Including the β-tert-butyl ester protection is thought to reduce this because of its bulkiness. This side reaction is, however, well documented in routine peptide synthesis even with side chain protection of aspartic acid. Incorporation of 0.1 M HOBt (hydroxybenzotriazole) in the deprotection solution has been shown to reduce aspartimide formation1,2. In many cases, however, this still leads to significant levels of aspartimide. The hexapeptide “VKDGYI” (SEQ ID NO: 2 has been shown to produce significant amounts of aspartimide related products during SPPS. This peptide was synthesized in a single-mode microwave manually with three 30-second 100 W cycles with ice bath cooling between each irradiation cycle. Maximum temperature was measured to be around 40° C. Significant aspartimide formation was detected using a 20% Piperidine in DMF solution. This was reduced with alterations in the deprotection solution, but only completely eliminated with use of an Hmb (hydroxyl-4-methoxybenzyl) dipeptide insertion of DG.
Table 1 illustrates the effect of deprotection reagent on aspartimide formation of VKDGYI (SEQ ID NO: 2)
Deprotection Reagent% Aspartimide20% Piperidine in DMF10.9020% Piperidine w/ 0.1M HOBt in DMF5.55Hexamethyleneimine/N-ethylpyrrolidine/HOBt in1.49NMP/DMSO20% Piperidine in DMF (w/HMB backbone protection)Not detected
Accordingly, given some of the practical disadvantages of piperidine and the potential side reactions, an opportunity exists for improving deprotection chemistry in SPPS.